Myxopyronin (Myx) is an ?-pyrone antibiotic that inhibits bacterial RNA polymerase (RNAP) through interactions with the RNAP switch region, a structural element that mediates conformational changes required for RNAP to bind and retain the DNA template in transcription. Myx does not inhibit eukaryotic RNAP I, RNAP II, or RNAP III. Myx exhibits potent antibacterial activity against Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Enterobacter cloacae, and Clostridium difficile in culture. Myx exhibits no cross-resistance with the inhibitors of bacterial RNAP in current clinical use in therapy of bacterial infection (the rifamycin antibacterial agents, rifampin, rifapentine, and rifabutin), and exhibits no or minimal cross-resistance with other inhibitors of bacterial RNAP under evaluation for future clinical use in therapy of bacterial infection. In preliminary work, we have shown that Myx functions by inhibiting bacterial RNAP through a binding site and mechanism that are different from those of rifamycin antibacterial agents. We have determined a crystal structure of a bacterial RNAP in complex with Myx, and we have constructed homology models of pathogen RNAP in complex with Myx. The crystal structure and homology models suggest alterations to the structure of Myx that are expected (i) specifically to increase potency against M. tuberculosis RNAP, exploiting a binding-site cysteine residue present in M. tuberculosis RNAP, or (ii) generally to increase potency against a broad spectrum of bacterial RNAP, exploiting a binding-site interfacial water molecule, and other structural features, present in a broad spectrum of bacterial RNAP. In further preliminary work, we have optimized procedures for total synthesis of Myx and Myx analogs, developed procedures for preparation of recombinant pathogen RNAP, and developed procedures for fluorescent and radiochemical assays of pathogen RNAP. We propose to leverage the mechanistic and structural information, synthetic procedures, and assay procedures developed in preliminary work in order to design, synthesize, and evaluate: (i) Myx analogs with increased efficacy against multidrug-resistant and extensively-drug-resistant M. tuberculosis, and (ii) Myx analogs with increased efficacy against a broad spectrum of drug-resistant pathogens. Analogs will be evaluated for inhibition of RNAP in vitro, for antibacterial activity in culture, and for cytotoxicity against mammalian cells in culture. Analogs of high promise will be evaluated for antibacterial activity in small-animal models of infection, and analogs of highest promise will be evaluated for bioavailability, pharmacokinetics, toxicity, and ability to scale synthesis. Primary target pathogens include: M. tuberculosis H37Rv and MDR/XDR, Staphylococcus aureus MSSA and MRSA, Enterococcus faecalis VSE and VRE, Streptococcus pneumoniae, Enterobacter cloacae, and Clostridium difficile. Drug-resistant bacterial infections are a major and growing threat. The proposed work is expected to provide two classes of new drug candidates: (1) antibacterial agents effective against multi-drug-resistant and extensively-drug-resistant tuberculosis, and (2) antibacterial agents effective against a broad spectrum of drug-resistant bacterial pathogens, including both public-health-relevant bacterial pathogens and biodefense-relevant bacterial pathogens.